Aging is the major risk factor for cardiovascular diseases (CVD). Two primary contributors to the increased risk of CVD with aging are stiffening of large elastic arteries and vascular endothelial dysfunction. The mechanisms responsible for these changes involve reductions in the bioavailability of the vascular-protective molecule, nitric oxide (NO), and the development of oxidative stress and inflammation. As such, establishing effective strategies that minimize and/or prevent these changes is a high biomedical priority. The nitrite anion (inorganic nitrite), once considered an inert byproduct of NO metabolism, is now recognized as a cytoprotective molecule that is a major storage form of NO with substantial therapeutic potential for the prevention and treatment of CVD. In old C57/BL6 mice we found that 3 weeks of sodium nitrite supplementation in drinking water reduced aortic pulse wave velocity (PWV), a measure of aortic stiffness, and intrinsic carotid artery stiffness by 50-80%, and completely restored NO-mediated endothelium-dependent dilation (EDD), a measure of vascular endothelial function. Nitrite treatment also reduced arterial oxidative stress and inflammation. The goal of the present R21 application is to translate our preclinical observations to humans. Using non- and minimally-invasive procedures, we will perform a pilot study to establish the efficacy of oral nitrite therapy for reducing large elastic artery stiffness and vascular endothelial dysfunction in middle-aged and older (MA/O) adults. We will also obtain initial insight into the mechanisms of these improvements with nitrite therapy. Hypothesis 1: Eight weeks of oral nitrite therapy (80 mg tablet x 2/d) vs. placebo (double-blind, randomized, cross-over design) will reduce large elastic artery stiffness ( aortic PWV, carotid artery compliance) and vascular endothelial dysfunction ( EDD, brachial artery flow-mediated dilation) in healthy adults aged 55-79 y. Hypothesis 2: The improvements in vascular function will be associated with evidence consistent with reduced oxidative stress and inflammation, and, perhaps, changes in structural protein markers and modulating factors. Impact on the Field. The biomedical significance of the proposed work includes: 1. Establishing the efficacy of a novel therapeutic agent (nitrite) for improving 2 important components of arterial aging and predictors of future CVD risk in MA/O adults; 2. Providing initial insight into possible mechanisms of action associated with reduced oxidative stress and inflammation, as well as changes in markers of structural protein and other modulatory factors; 3. Generating preliminary data that will provide the experimental basis for performing a larger, more comprehensive randomized clinical trial (future R01) that will: determine the effects of oral nitrite therapy on additional primary outcomes; obtain more detailed, cause-and-effect evidence on cellular and molecular mechanisms of action; extend efficacy of nitrite therapy to MA/O adults with major CVD risk factors and/or clinical CVD. PUBLIC HEALTH RELEVANCE: The proposed research will determine the effectiveness of sodium nitrite, a naturally occurring compound in the body, for improving the health and function of arteries in middle-aged and older adults. The study also will provide insight into how sodium nitrite therapy improves artery health by determining the physiological mechanisms (biological reasons) involved. Overall, the proposed research will provide important new scientific evidence on the effectiveness of sodium nitrite for decreasing the risk of developing cardiovascular diseases with aging.